INTERACTION OF PHOSPHORYLATED TRYPTOPHAN-HYDROXYLASE WITH 14-3-3 PROTEINS

Rabbit brain tryptophan hydroxylase (TPH) has been expressed in insect cells (Spodoptera frugiperda) as a histidine-tagged enzyme. The speci fic activity of the purified fusion enzyme is 80 nmol of 5-hydroxytryp tophan/min/mg. Multifunctional regulatory 14-3-3 proteins were purifie d from fresh bovine brain. Phosphorylation and 14-3-3 proteins play im portant roles in the regulation of TPH activity. We have found that ph osphorylation of TPH by cAMP-dependent protein kinase increased the ac tivity of the hydroxylase by 25-30% and that 14-3-3 proteins increased the hydroxylase activity of phosphorylated TPH by similar to 45%. Und er these conditions, the 14-3-3 proteins were not phosphorylated, and unphosphorylated TPH was not activated by 14-3-3 proteins. Surface pla smon resonance analysis demonstrated that 14-3-3 proteins bind to phos phorylated TPH with an affinity constant (K-a) of 4.5 x 10(7) M-1. Bin ding studies using affinity chromatography also showed that 14-3-3 pro teins interact with phosphorylated TPH. The dephosphorylation of TPH b y protein phosphatase-l was inhibited by 14-3-3 proteins. Our results demonstrate that 14-3-3 proteins form a complex with phosphorylated br ain TPH, thereby increasing its enzymatic activity and inhibiting its dephosphorylation.